Citrate Utilization Test- Principle, Procedure, Results, Uses

What is Citrate Utilization Test?

The citrate utilization test is a part of the IMViC test (Indole, Methyl Red, Vogues-Proskauer, and Citrate Test) that differentiates organisms on the basis of their ability to use citrate as a sole source of energy.

• The citrate test is performed along with the other IMViC tests to differentiate Gram-negative bacilli of the Enterobacteriaceae family.
• It is an important test that allows the species-level identification of the members of the Enterobacteriaceae family.
• With the exception of a few species, Salmonella, Edwardsiella, Citrobacter, Klebsiella, Enterobacter, Serratia, and Providencia usually give a positive reaction, and Escherichia, Shigella, Morganella, and Yersinia give a negative reaction. Proteus is a citrate variable.
• The test is also called Simmon’s citrate test as it utilizes Simmon’s citrate agar that contains citrate as the major source of energy.
• The medium further contains ammonium hydrogen phosphate (NH₄H₂PO₄) as the sole source of nitrogen.
• Organisms that give a positive result in this test should be capable of fermenting citrate in the presence of the enzyme citrase.

Principle of Citrate Utilization Test

• Citrate agar is used to test the ability of an organism to utilize citrate as a source of energy.
• The agar medium contains citrate as the sole carbon source and inorganic ammonium salts as the sole source of nitrogen.
• The growth of the organism is indicative of the utilization of citrate as it is an intermediate metabolite in the Krebs cycle. The enzyme citrase breaks down citrate into oxaloacetate and acetate, where oxaloacetate is further broken down to form pyruvate and carbon dioxide.
• The release of carbon dioxide induces the metabolism of ammonium salts, causing the formation of ammonia or sodium carbonate, both of which increase the alkalinity of the medium.
• The shift in pH turns the bromthymol blue indicator in the medium from green to blue above pH 7.6

Citrate → Oxaloacetic acid → Pyruvic acid + CO₂

Excess of sodium from sodium citrate + CO₂ +H₂O → Na₂CO₃

• The growth of the organism on the medium followed by the change in the color; as a result, of citrate metabolism gives a positive citrate test.

Microorganisms Tested

Members of Enterobacteriaceae, as part of the identification to the species level

Media, Reagents, and Supplies Used

Media Used

• Simmon’s Citrate agar is used as the medium to test the ability of an organism to utilize citrate as a sole source of energy.
• Simmon’s Citrate agar is sold commercially by different vendors in the form of dehydrated powder. However, it can also be prepared in the laboratory if the necessary ingredients are available.
• The following is the composition of Simmon’s Citrate Agar:

Supplies Used

• Inoculating loops or needles
• Incubator at 35-37°C

Procedure of Citrate Utilization Test

1. Preparation of the media

• In a beaker, 24.28 grams of the dehydrated powder or lab-prepared media is added to 1000 milliliters of pure distilled or deionized water.
• The solution is then heated to bring it to a boil in order to dissolve the medium completely.
• The dissolved medium is then dispensed into tubes and sterilized in an autoclave at 15 lbs pressure (121°C) for 15 minutes.
• Once the autoclaving process is complete, the tubes are taken out and cooled at a slanted position to a temperature of about 40-45°C. The position should be maintained in order to obtain butts of 1.5 – 2.0 cm depth.

2. Utilization test

• A well-isolated colony is taken from an 18-24 hour culture with a sterile inoculating needle.
• The citrate agar tubes are inoculated by streaking the surface of the slant. The slant should be streaked back and forth with the loop or the inoculating stick.
• The cap of the test tubes should be left loosened to ensure adequate aeration.
• The tubes are then incubated aerobically at 35-37°C for up to 4 days.
• The test tubes should be examined daily for 4 days before discarding the result as a negative.
• The change in color, if present, is observed.

Quality Control

• Aseptic Simmon’s Citrate Agar should appear as forest green colored slightly opalescent gel slants.
• The agar should be inspected for evidence of freezing, contamination, cracks, dehydration, and bubbles prior to use. Any tubes that might appear blue before use should be discarded.
• The positive and negative control for the test are:

Klebsiella pneumoniae: Citrate Positive- Positive Control
Escherichia coli: Citrate Negative; Negative Control

Result Interpretation of Citrate Utilization Test

Figure: Citrate Utilization Test. Image Source: Bailey and Scott’s Diagnostic Microbiology. Elsevier.

• A positive test is demonstrated by growth with a color change from green to intense blue along the slant.
• A negative test is demonstrated by no growth and no color change, and the color of the slant remains green.

The following table demonstrates the result of Citrate utilization of important medical bacteria:

Uses of Citrate Utilization Test

• Citrate test is used to determine the ability of an organism to utilize citrate as a sole source of energy.
• A citrate test is performed as a part of the IMViC test to differentiate members of Enterobacteriaceae.
• The test is also used for the differentiation between fecal coli and members of the aerogenes group on the basis of citrate utilization.

Limitations of Citrate Utilization Test

• Luxuriant growth on the slant without an accompanying color change may indicate a positive test. However, if the agar does not turn blue on further incubation, the test should be repeated with a smaller inoculum.
• Tests with equivocal results should be repeated.
• The slant should not be stabbed since the test requires an aerobic environment.
• Inoculation should not be done from broth cultures, due to the risk of carryover of media.
• To avoid false-positive reactions, a light inoculum should be used in order to prevent the carryover of substances from previous media.
• The reactions of this medium alone are not sufficient for identification to the species level, and further tests should be performed for confirmation.

References and Sources

• Simmons Citrate Agar. M099S. HiMedia Laboratories.
• Biochemical Tests for the Identification of Aerobic Bacteria. (2016). Clinical Microbiology Procedures Handbook, 3.17.1.1–3.17.48.3. DOI:10.1128/9781555818814.ch3.17.1.
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